The myosin II complex is involved in the regulation of eukaryotic cell structure and motility. Cell structure modulation in pathophysiology can result in modulation of gaps between cells in tissues, such as epithelial or endothelial layers that form tissue barriers, or in modulation of cell motility or chemotoxis, such as in the metastasis of tumor cells. Activation of the myosin II complex in physiology and pathophysiology requires phosphorylation of the myosin regulatory light chains (MLC) by myosin light chain kinase (MLCK), a highly specialized calcium/CaM regulated protein kinase that has MLC as its only physiological substrate. [T. J. Lukas, S. Mirzoeva and D. M. Watterson in (Van Eldik, L. J. and Watterson, D. M., eds.) Calmodulin and signal transduction, Academic Press 1998, pp. 65-168.] There is more than multiple MLCK enzymes, but the gene products that are most widely viewed as critical to disease and potential therapeutic intervention are the forms from one genetic locus. The forms are sometimes referred to as smooth muscle MLCK for the shorter form, due to its historical discovery first in smooth muscle tissue, and the long form or MLCK210 (in reference to the MW of 210,000 for the vertebrate protein, and often implied when the terms endothelial, epithelial, or non-muscle MLCK are used. [T. J. Lukas, S. Mirzoeva and D. M. Watterson in (Van Eldik, L. J. and Watterson, D. M., eds.) Calmodulin and signal transduction, Academic Press 1998, pp. 65-168.]. The predominant isoform of MLCK involved in tissue barrier regulation and tumor cell metastasis is the long form, or MLCK210. [A. D. Verin, V. Lazar, R. J. Torry, C. A. Labarrere, C. E. Patterson and J. G. Garcia, Am. J. Respir. Cell. Mol. Biol. 19 (1998) 758-66; D. M. Watterson, M. Collinge, T. J. Lukas, L. J. Van Eldik, K. G. Birukov, O. V. Stepanova and V. P. Shirinsky, FEBS Lett 373 (1995) 217-20.] A direct demonstration of the in vivo importance of MLCK210 in pathophysiology and its potential as a therapeutic target comes from the analysis of mice in which the gene for MLCK210 has been selectively knocked out, leaving the short form of MLCK intact. MLCK210 knockout (KO) mice, for example, are protected from inflammation linked tissue injury and death. The MLCK KO mice are protected in an animal model of acute immune-mediated diarrhea disease (e.g., Crohn's disease and episodic diarrhea associated with other diseases), in microvascular and lung injury associated with sepsis and microbial toxin induced injury, in neurologic outcomes from blood-brain barrier dysfunction associated with brain injury or disease, and in death from severe burn as a result of multi-organ failure. Congruent with the MLCK210 KO mouse results, wild type mice subjected to such tissue and microvascular injuries are protected by treatment with a selective MLCK inhibitor. In addition to providing a well established link between MLCK and tissue barrier functions that are points of susceptibility in disease progression in inflammation related injuries, these studies with MLCK210 KO mice and therapeutic doses of MLCK inhibitors established MLCK as a potential non-immune therapeutic target for certain inflammation related disorders. Similarly, therapeutic doses of MLCK inhibitors that inhibit tumor cell chemotaxis provides a link between the motility function of MLCK as a target in cancer and other disorders. As addressed elsewhere herein, it is understood in the art that MLCK affects tissue barrier function, cell motility and/or adhesion.
Currently, therapeutic approaches in diseases such as inflammatory bowel disorders or complications of severe bacterial infection are focused on the cytokines which are up-regulated and are among the active mediators of both endothelial and epithelial barrier dysfunction. Antibody therapies targeting these cytokines have been used, but they have limited stability, restricted utility due to their limited tissue distribution, and are expensive protein therapeutics with individual sets of safety problems. Non-protein therapies involving small molecules such as statins and glucocorticoids have some clinical effectiveness for inflammatory conditions, but are limited by their multiple biological effects and toxicity. In cancer chemotherapy, existing therapies are focused on cytotoxicity of the tumor and not on the motility related functions involved in metastasis. Accordingly, there is an unmet need for new and safe compounds, to optionally use as a co-therapy with other drugs or mechanical devices, to attenuate barrier and/or cell motility associated dysfunctions.
Based on the demonstrated protection from epithelial and endothelial barrier dysfunction afforded by inhibition of MLCK, by gene knockout or use of kinase inhibitors, one approach has been small molecule inhibitors of MLCK. However, currently available inhibitors are peptidic in nature or are expensive bioavailable small molecules for chemical biology research that are not readily amenable to further development. Accordingly, there remains an ongoing search in the art for a class of readily available, small molecule compounds, with corresponding system mechanism(s) of intervention, for MLCK inhibition.